This invention relates to nucleic acid and amino acid sequences of human GTPase-associated proteins and to the use of these sequences in the diagnosis, treatment, and prevention of cell proliferation disorders, autoimmune/inflammatory disorders, and vesicle trafficking disorders.
The GTPase superfamily includes many proteins which act as intracellular switches in signal transduction and vesicle trafficking. (Lodish, H. et al. (1995) Molecular Cell Biology, Scientific American Books, Inc., New York, N.Y., pp. 876-877.) The family includes both heterotrimeric GTPases (G proteins) and low molecular weight GTPases related to Ras. In both cases the GTPase activity is regulated through interactions with other proteins.
The heterotrimeric G proteins, a family of peripheral membrane GTPases, are present in all cells. They control metabolic, humoral, neural, and developmental functions by transducing hormonal, neurotransmitter, and sensory signals into an array of cellular responses. Each G protein is composed of an alpha (xcex1), a beta (xcex2), and a gamma (xcex3) subunit, associated as a complex in an inactive GDP-bound form. Gxcex1 binds GDP or GTP and contains the GTPase activity. The xcex2xcex3 complex enhances binding of Gxcex1 to a receptor. Gxcex3 is necessary for the folding and activity of Gxcex2. (Neer, E.J. et al. (1994) Nature 371:297-300.) Multiple homologs of each subunit have been identified in mammalian tissues, and different combinations of subunits have specific functions and tissue specificities. (Spiegel, A. M. (1997) J. Inher. Metab. Dis. 20:113-121.)
G protein activity is triggered by seven-transmembrane cell surface receptors (G protein coupled receptors; GPCRs) which respond to lipid analogs, amino acids and their derivatives, peptides, cytokines, and specialized stimuli such as light, taste, and odor. Activation of the receptor by its stimulus causes the replacement of the G protein-bound GDP with GTP. Gxcex1-GTP dissociates from the receptor and the xcex2xcex3 complex. Both Gxcex1-GTP and the xcex2xcex3 complex stimulate or inhibit effector molecules to transmit the signal delivered to the GPCR. The signaling is stopped when Gxcex1 hydrolyzes its bound GTP to GDP and reassociates with the xcex2xcex3 complex. G60  interacts with the effectors adenylate cyclase, ion channels, and phospholipase C-xcex2, and the xcex2xcex3 complex interacts with adenylate cyclase, phospholipase C-xcex2, xcex2-adrenergic receptor kinase, phosducin, phosphoinositide 3-kinase, and potassium channels. In yeast, the xcex2xcex3 complex mediates a G protein dependent mating response. (Mxc3xcller, S. et al. (1996) J. Biol. Chem. 271:11781-11786; Meij, J. T. A. (1996) Mol. Cell. Biochem. 157:31-38; and Watson, A. J. et al. (1994) J. Biol. Chem. 269:22150-22156.)
Gxcex2 proteins, also known as xcex2 transducins, are all about 340 amino acids in length and contain seven tandem repeats of the WD-repeat sequence motif, a motif found in many proteins with regulatory functions. WD-repeat proteins contain from four to eight copies of a loosely conserved repeat of approximately 40 amino acids. Each repeat contains a central conserved region bracketed by Gly-His and Trp-Asp (WD) residues. The three-dimensional structure of the xcex2xcex3 complex has been solved, and Gxcex2 was shown to fold into a circular seven-bladed xcex2 propeller. Other WD-repeat proteins are likely to form similar xcex2 propeller structures. (Neer, supra; and Garcia-Higuera, I. et al. (1996) Biochemistry 35:13985-13994.)
Other proteins with seven WD repeats have been found, some of which have roles in signal transduction. Mutations in LIS1, a subunit of the human platelet activating factor acetylhydrolase, cause Miller-Dieker lissencephaly, a severe brain malformation. MSI1 is a negative regulator of Ras-mediated cAMP increase in yeast. RACK1 binds activated protein kinase C, and RbAp48 binds retinoblastoma protein. CstF is required for polyadenylation of mammalian pre-mRNA in vitro and associates with subunits of cleavage-stimulating factor. xcex2Trcp is a yeast protein whose expression suppresses mutations in CDC15, a gene needed for normal anaphase in yeast. Uncharacterized 7 WD-repeat proteins exist as well. (Neer, supra.)
Research indicates that an irregularity in any GPCR pathway component can cause a physiological defect. (Meij, supra.) For example, mutations in the components of the cell signaling cascade as well as alterations in the expression pattern of these components may result in abnormal activation of leukocytes and lymphocytes, leading to the tissue damage and destruction seen in autoimmune diseases such as rheumatoid arthritis, biliary cirrhosis, hemolytic anemia, lupus erythematosus, and thyroiditis. T cell activation is a G protein regulated process. (Aussel, C. et al. (1988) J. Immunol. 140:215-220.)
Irregularities in G protein signaling also have a role in abnormal cell proliferation. Cyclic AMP stimulation of brain, thyroid, adrenal, and gonadal tissue proliferation is regulated by G proteins. Mutations in Gxcex1 subunits have been found in growth-hormone-secreting pituitary somatotroph tumors, hyperfunctioning thyroid adenomas, and ovarian and adrenal neoplasms. (Spiegel, supra.)
Other genetic disorders caused by loss or gain of function mutations in Gxcex1 subunits include Albright hereditary osteodystrophy, pseudohypoparathyroidism type Ia with precocious puberty, McCune-Albright syndrome, and congenital night blindness. (Spiegel, supra.) GPCR mutations are responsible for many diseases including color blindness, retinitis pigmentosa, congenital night blindness, nephrogenic diabetes insipidus, familial adrenocorticotropic hormone (ACTH) resistance, hypergonadotropic ovarian dysgenesis, familial male precocious puberty, male pseudohermaphroditism, sporadic hyperfunctional thyroid nodules, familial nonautoimmune hyperthyroidism, familial hypothyroidism, familial hypocalciuric hypercalcemia/neonatal severe primary hyperparathyroidism, familial hypoparathyroidism, congenital bleeding, Hirschsprung disease, Jansen metaphyseal chondrodysplasia, and familial growth hormone deficiency. (Spiegel, supra.) A G-protein controlled pathway, the xcex2-adrenoreceptor/adenylate cyclase pathway, appears to be desensitized in heart failure. (Meij, supra.)
Rab proteins, which are Ras-related low molecular weight GTPases, regulate vesicular transport between subcellular compartments of eukaryotic cells. During this process, vesicles bud from a donor membrane and fuse with a recipient to deliver internalized materials. Rab proteins assist the binding of transport vesicles to their accepter organelles and initiate the vesicle fusion process using the energy from GTP hydrolysis. More than 30 Rab proteins have been identified in a variety of species, and each has a characteristic intracellular location and distinct transport function. Rab5 is localized to endosomes and regulates the fusion of early endosomes into late endosomes. Rab5 function in vesicular transport requires the cooperation of other proteins, including Rab-escort proteins, Rab-specific guanine nucleotide dissociation inhibitor, and rabaptins. (Vitale, G. et al. (1995) Cold Spring Harbor Symp. Quant. Biol. 60:211-220; and Vitale, G. et al. (1998) EMBO J. 17:1941-1951.) Several putative Rab5-interacting proteins expressed in human HeLa cells have been identified using a yeast two-hybrid screen, and their partial nucleotide and amino acids sequences determined. (Vitale (1995) supra.)
Defects in protein trafficking to organelles or the cell surface are involved in numerous human diseases and disorders. Defects in the trafficking of membrane-bound receptors and ion channels are associated with cystic fibrosis (cystic fibrosis transmembrane conductance regulator), glucose-galactose malabsorption syndrome (Na+/glucose cotransporter), hypercholesterolemia (low-density lipoprotein receptor), and forms of diabetes mellitus (insulin receptor). Abnormal hormonal secretion is linked to disorders including diabetes insipidus (vasopressin), hyper- and hypoglycemia (insulin, glucagon), Grave""s disease and goiter (thyroid hormone), and Cushing""s and Addison""s diseases (ACTH).
Cancer cells secrete excessive amounts of hormones or other biologically active peptides. Disorders related to this excessive secretion include: fasting hypoglycemia due to increased insulin secretion from insulinoma-islet cell tumors; hypertension due to increased epinephrine and norepinephrine secreted from pheochromocytomas of the adrenal medulla and sympathetic paraganglia; and carcinoid syndrome, which includes abdominal cramps, diarrhea, and valvular heart disease, caused by excessive amounts of vasoactive substances (serotonin, bradykinin, histamine, prostaglandins, and polypeptide hormones) secreted from intestinal tumors. Ectopic synthesis and secretion of biologically active peptides includes ACTH and vasopressin in lung and pancreatic cancers, parathyroid hormone in lung and bladder cancers, calcitonin in lung and breast cancers, and thyroid-stimulating hormone in medullary thyroid carcinoma.
The discovery of new human GTPase-associated proteins and the polynucleotides encoding them satisfies a need in the art by providing new compositions which are useful in the diagnosis, treatment, and prevention of cell proliferation disorders, autoimmune/inflammatory disorders, and vesicle trafficking disorders.
The invention features substantially purified polypeptides, human GTPase- associated proteins, referred to collectively as xe2x80x9cGPAPxe2x80x9d and individually as xe2x80x9cGPAP-1xe2x80x9d and xe2x80x9cGPAP-2.xe2x80x9d In one aspect, the invention provides a substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3.
The invention further provides a substantially purified variant having at least 90% amino acid identity to the amino acid sequences of SEQ ID NO:1 or SEQ ID NO:3, or to a fragment of either of these sequences. The invention also provides an isolated and purified polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3. The invention also includes an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3.
Additionally, the invention provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3, as well as an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3.
The invention also provides an isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, a fragment of SEQ ID NO:2, and a fragment of SEQ ID NO:4. The invention further provides an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide sequence comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, a fragment of SEQ ID NO:2, and a fragment of SEQ ID NO:4, as well as an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, a fragment of SEQ ID NO:2, and a fragment of SEQ ID NO:4.
The invention further provides an expression vector containing at least a fragment of the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3. In another aspect, the expression vector is contained within a host cell.
The invention also provides a method for producing a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3, the method comprising the steps of: (a) culturing the host cell containing an expression vector containing at least a fragment of a polynucleotide encoding the polypeptide under conditions suitable for the expression of the polypeptide; and (b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3 in conjunction with a suitable pharmaceutical carrier.
The invention further includes a purified antibody which binds to a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3, as well as a purified agonist and a purified antagonist to the polypeptide. The invention also provides a method for treating or preventing a cell proliferation disorder associated with the decreased expression or activity of GPAP, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a substantially purified polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3.
The invention also provides a method for treating or preventing a cell proliferation disorder associated with the increased expression or activity of a GPAP, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of the polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3.
The invention also provides a method for treating or preventing an autoimmune/inflammatory disorder associated with the decreased expression of activity of a GPAP, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a substantially purified polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3.
The invention also provides a method for treating or preventing an autoimmune/inflammatory disorder associated with the increased expression or activity of a GPAP, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of the polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3.
The invention also provides a method for treating or preventing a vesicle trafficking disorder, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a substantially purified polypeptide having an amino acid sequence of SEQ ID NO:3 or a fragment of SEQ ID NO:3.
The invention also provides a method for detecting a polynucleotide encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3 in a biological sample containing nucleic acids, the method comprising the steps of: (a) hybridizing the complement of the polynucleotide sequence encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3 to at least one of the nucleic acids of the biological sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide encoding the polypeptide in the biological sample. In one aspect, the nucleic acids of the biological sample are amplified by the polymerase chain reaction prior to the hybridizing step.